Paving the Way for C₄ Evolution: Study of C₃-C₄ Intermediate Species in Grasses

C₄ photosynthesis originated as an adaptation to low atmospheric CO₂ concentrations 30–35 million years ago (Sage, 2016). Although accounting for only 3% of the vascular plants on earth, C₄ plants contribute 25% of terrestrial photosynthesis (Westhoff and Gowik, 2010), thus providing a highly desirable system to increase carbon fixation efficiency and enhance crop yield. The transition from C₃ to C₄ photosynthesis has occurred more than 62 times independently in a complex process with a combination of anatomical, biochemical, and physiological changes in leaves. In spite of their multiple origins, C₄ plants are only present in certain angiosperm families, including about 5,000 grasses, 1,300 sedges, and 1,800 dicot species, indicating developmental and anatomical constraints for C₄ evolution (Sage, 2016).

In C₄ photosynthesis, CO₂ is initially taken up by mesophyll cells and converted into a four-carbon (C₄) compound by phosphoenolpyruvate carboxylase (PEPC), which has high affinity for CO₂. Then, the C₄ compound is transported to the chloroplasts of the bundle sheath cells where it releases CO₂, resulting in increased CO₂ concentration and enhanced carboxylase activity of Rubisco, the rate-limiting enzyme of photosynthesis. The mesophyll and bundle sheath are arranged in a “wreath-like” shape surrounding the vascular bundle, a structure referred to as the Kranz anatomy. In grasses and sedges, there are two layers of sheath cells, an inner bundle sheath (also called mestome sheath) and an outer bundle sheath. One or both types of sheath cells may be co-opted for C₄ function, although one of them is often lost during C₄ evolution (Lundgren et al., 2014).

A few anatomical and cellular adaptations are required for efficient C₄ physiology: (1) compartmentation of CO₂ assimilation and fixation by increasing PEPC activity in the mesophyll cells and limiting Rubisco only to bundle sheath chloroplasts; (2) increased size and number of chloroplasts in the bundle sheath; (3) shortened distance between mesophyll and bundle sheath cells by increasing vein density and/or enlarging bundle sheath and mesophyll cell size; and (4) enrichment of plasmodesmata between mesophyll and bundle sheath cells to facilitate metabolic exchange (Fig. 1; Lundgren et al., 2014; Sage, 2016). If the evolution of these traits is stepwise or progressive, we may expect to observe species with intermediate traits between C₃ and C₄ during evolutionary history. Indeed, C₂ photosynthesis that utilizes Gly (two-carbon compound [C₂]) as a shuttle to concentrate CO₂ is proposed as an intermediate state during C₄ evolution. It is worth noting that although grasses and sedges dominate C₄ species, <20% of known intermediate species are monocots, hindering our understanding of C₄ evolution within monocots (Sage et al., 2014). In this issue of Plant Physiology, Khoshravesh et al. (2020) compare various anatomical, ultrastructural, and biochemical traits in nine species of the subtribe Neurachninae of the grass family, and identified new C₃-C₄ intermediate species.

Figure 1.

A simplified diagram comparing C₃, C₂ and C₄ anatomy and biochemical reactions. In C₃ plants, photosynthesis and photorespiration occur in both mesophyll and bundle sheath (BS) cells. CO₂ assimilation and fixation occur within both cell types. In C₂ plants, photosynthesis occurs in both mesophyll and BS cells. GDC (G in the diagram) is only present in the mitochondria of BS cells. Gly (C₂) produced in mesophyll cells is transported into the BS, where CO₂ is released by GDC for carbon fixation. In C₄ plants, Rubisco (labeled R in the diagram) is lost in the chloroplast of mesophyll cells. CO₂ is converted into C₄ compound in mesophyll cells, and the latter is transported into BS cells for carbon fixation. Figure by Yunqing Yu.

C₂ photosynthesis differs from C₄ in biochemical reactions in that C₂ plants concentrate CO₂ by limiting the photorespiratory reactions in the mitochondria of bundle sheath cells. Gly generated by oxygenation reaction of Rubisco in mesophyll cells is transported to the bundle sheath cells and then converted to Ser and CO₂ by the mitochondrial Gly decarboxylase complex (GDC; Fig. 1). Therefore, a major feature of C₂ anatomy is increased abundance of mitochondria with high GDC activity, and aggregation of mitochondria and chloroplasts in the inner side of bundle sheath cells to minimize gas exchange with mesophyll cells (Fig. 1). The close association between mitochondria and chloroplasts also facilitates diffusion of photorespired CO₂ into the chloroplasts, and thus concentrates CO₂ surrounding Rubisco (Sage et al., 2014). C₂ anatomy exhibits several C₄-like features (although less pronounced than C₄), including prominent bundle sheath cells, increased vein density, more abundant and asymmetrically distributed chloroplasts in the bundle sheath, and higher plasmodesmatal frequency (Fig. 1; Sage et al., 2014; Khoshravesh et al., 2020).

In addition to typical C₂ photosynthesis, other C₃-C₄ intermediate species with different intermediate traits may shed light on C₄ evolution. Khoshravesh et al. (2020) focused on subtribe Neurachninae, which contains a well-known C₂ species (Neurachne minor), two C₄ species (Neurachne munroi and Neurachne muelleri), and several putative C₃ species (Neurachne alopecuroidea, Neurachne queenslandica, Thyridolepis mitchelliana, Neurachne tenuifolia, Neurachne annularis, and Neurachne lanigera). The Neurachne species possess two layers of bundle sheath, an inner suberized mestome sheath where carbon fixation occurs (functional bundle sheath), and an outer parenchymatous sheath that separates mesophyll cells and mestome sheath. Khoshravesh et al. (2020) measured C* (the CO₂ compensation point) as an indicator of photorespiratory CO₂ refixation. They further comprehensively quantified traits including mestome sheath and mesophyll cell size in three dimensions, the ratio of different cell types, mitochondria and chloroplast number and size, vein spacing, plasmodesmatal frequency, and the abundance of Gly decarboxylase subunit P (GLDP) and Rubisco large subunit by immunolabeling. These analyses revealed a linear relationship between the above traits and C*, indicating the importance of these anatomical and biochemical characters contributing to photosynthetic efficiency.

Furthermore, among the putative C₃ species, N. lanigera and N. annularis exhibited various degrees of intermediate traits between typical C₃ and C₂. For example, compared to typical C₃ species, N. lanigera has significantly shorter and wider mestome sheath cells, increased number of chloroplasts and mitochondria in the mestome sheath, greater GLDP labeling, and enriched plasmodesmata. Consistently, C* of N. lanigera is significantly lower than typical C₃ species particularly at higher temperature (35°C), although it is still higher than C* in the C₂ species N. minor. In contrast, N. annularis functions as a C₃, as its C* is similar to that of other C₃ species. However, it shows subtle C₂-like characteristics, including slightly wider mestome sheath cells and greater GLDP abundance per mestome sheath chloroplast, indicating its early stage in C₂ evolution (Khoshravesh et al., 2020).

Unlike previous phylogenetic analyses showing that C₃-C₄ species indeed represent an intermediate state in C₄ evolution (Schlüter and Weber, 2016), the progressive C₄ traits of the Neurachne species do not reflect the phylogenetic relationships at the species level. In other words, an ancestral state with C₃-C₄ intermediacy may lead to the evolution of C₄, C₂,or even C₃ species, suggesting that C₃ and C₃-C₄ intermediates may represent partial reversals, having evolved from a C₄-like ancestor. Analyses of more tribes or clades with multiple C₃-C₄ species may further advance our understanding of the direction of C₃/C₄ evolution.